Abstract 5344: High-throughput CRISPR-Cas9 knockout screens identify loss of <i>miRNA1304-5p</i> targeting the RAS/MAPK pathway as a modulator of resistance to ALK inhibitors in high-risk neuroblastoma
Authors: Perla Pucci, Liam Lee, Suzanne Turner, Leila Jahangiri, Jamie Matthews, Amos Burke, Patrick Reynolds, Lukas Kenner, Olaf Merkel, Fikret Rifatbegovic, Ji Luo, Peter Ambros, MiaoJun Han
Journal: Cancer Research (2022)

Abstract Anaplastic lymphoma kinase (ALK)-targeted therapy is a promising strategy for the treatment of ALK-positive neuroblastoma (NB). Several ALK tyrosine kinase inhibitors (ALK TKIs) have been approved for the treatment of Non-Small Cell Lung Carcinoma (NSCLC) and are currently in clinical trials for NB therapy. Recent evidence reported that ALK TKI resistance can occur in cancer patients, making the long-term efficacy of these therapies challenging. With the use of high-throughput and genome-wide CRISPR-Cas9 knockout screens, we identified loss of genes associated with decreased sensitivity to ALK TKIs in ALK-positive NB cells. Specifically, we discovered that CRISPR-Cas9 mediated knockout of miR-1304-5p desensitises ALK-positive NB to the ALK TKIs brigatinib and ceritinib. Inhibition of miR-1304-5p decreased, while mimics increased the sensitivity of NB cells to these ALK TKIs. Furthermore, we showed that miR-1304-5p targets the RAS/MAPK pathway, thereby decreasing cell viability via induction of apoptosis. Among the targets of miR-1304-5p, NRAS expression levels are of prognostic significance to NB patients suggesting it may drive an aggressive disease phenotype. Indeed, a combination treatment applying ALK TKIs and miR-1304-5p mimics increased treatment response. A similar effect was achieved using RAS pharmacological inhibition, which resulted in synergistic activity in MYCN Wild Type (WT) but had less effect on MYCN-amplified cells, suggesting that this pathway could be differentially regulated according to MYCN status. miR-1304-5p and NRAS expression, in patient-derived xenografts (PDXs) of high-risk NB harbouring ALK mutations, confirmed the relevance of this regulatory axis in NB and suggested that the reduced ALK TKI response in MYCN-positive PDXs could be due to inhibition of miR-1304-5p, and the combination of ALK and NRAS inhibition showed extraordinary anti-tumor efficacy compared to single agents. These findings suggest that the miR-1304-5p/NRAS axis alters the sensitivity of NB to ALK inhibitors and suggest that the modulation of this pathway in combination with ALK inhibition is a promising approach to improve NB treatment response and ultimately patient survival. Citation Format: Perla Pucci, Liam Lee, Suzanne Turner, Leila Jahangiri, Jamie Matthews, Amos Burke, Patrick Reynolds, Lukas Kenner, Olaf Merkel, Fikret Rifatbegovic, Ji Luo, Peter Ambros, MiaoJun Han. High-throughput CRISPR-Cas9 knockout screens identify loss of miRNA1304-5p targeting the RAS/MAPK pathway as a modulator of resistance to ALK inhibitors in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5344.